The overall goal of this proposal is to further validate dUTPase as a target for drug discovery. dUTPase catalyzes the hydrolysis of dUTP to form dUMP and PPi. This reaction effectively removes dUTP from the DNA biosynthetic pathway, thereby preventing detrimental uracil misincorporation into DNA. Broadly utilized inhibitors of thymidylate metabolism (i.e. 5-FU and methotrexate) induce a severe depletion of TTP pools and in some cases, a concurrent accumulation of dUTP pools that results in the iterative misincorporation of uracil into DNA, leading to DNA damage and cell death. Although some cancer cell lines are able to accumulate dUTP pools upon TS inhibition, others do not; leading to the hypothesis that elevated dUTPase activity in tumors prevents dUTP accumulation resulting in drug resistance. Indeed, overexpression of dUTPase in tumor specimens is associated with non-response to TS-directed chemotherapy in metastatic colon cancer (Ladner, et al. 2000). dUTPase, the key regulator of dUTP pools, represents a promising alternative therapeutic target within a pathway of proven clinical utility. Inhibition of dUTPase in combination with traditional agents may provide a novel approach to fully exploit thymidylate biosynthesis as a chemotherapeutic target. We hypothesize that an effective dUTPase inhibitor will enhance the efficacy of traditional TS inhibitors and overcome drug resistance that results from overexpression of intratumoral dUTPase. The main objective of this proposal is to down regulate dUTPase expression in a series of cancer cell lines and validate the impact of inhibited activity on the efficacy of agents that target thymidylate biosynthesis. Specific Aim 1: Does lowering cellular dUTPase activity sensitize human cancer cells to widely utilized inhibitors of thymidyate and folate metabolism? Specific Aim 1 investigates the effect of dUTPase down regulation on the toxicity of chemotherapeutic agents that inhibit thymidylate synthase and folate metabolism. Our approach utilizes siRNA-mediated down regulation of dUTPase activity, and preliminary data demonstrating our ability to utilize this technique is presented. Specific Aim 2: Does the synergy between TS inhibitors and lowered dUTPase activity correlate with the biochemical endpoints of uracil misincorporation? A mechanistic analysis will be used to evaluate the biochemical enpoints underlying the uracil misincorporation pathway.